U.S. patent number 10,261,096 [Application Number 15/421,838] was granted by the patent office on 2019-04-16 for method and kit for diagnosing depression after acute coronary syndrome using homocysteine.
This patent grant is currently assigned to INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY. The grantee listed for this patent is INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY. Invention is credited to Hee Ju Kang, Jae Min Kim.
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United States Patent |
10,261,096 |
Kim , et al. |
April 16, 2019 |
Method and kit for diagnosing depression after acute coronary
syndrome using homocysteine
Abstract
This invention relates to a method of diagnosing the depression,
and more particularly to a method and kit for diagnosing depression
after acute coronary syndrome, by measuring the concentration of a
specific biomarker in the blood of a patient suffering from acute
coronary syndrome to predict depression after acute coronary
syndrome.
Inventors: |
Kim; Jae Min (Gwangju,
KR), Kang; Hee Ju (Gwangju, KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY |
Gwangju |
N/A |
KR |
|
|
Assignee: |
INDUSTRY FOUNDATION OF CHONNAM
NATIONAL UNIVERSITY (Gwangju, KR)
|
Family
ID: |
61242221 |
Appl.
No.: |
15/421,838 |
Filed: |
February 1, 2017 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180059121 A1 |
Mar 1, 2018 |
|
Foreign Application Priority Data
|
|
|
|
|
Aug 30, 2016 [KR] |
|
|
10-2016-0110860 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/6893 (20130101); G01N 33/6815 (20130101); B01D
15/08 (20130101); C12Q 1/6883 (20130101); G01N
2800/304 (20130101); C12Q 2600/156 (20130101); C12Q
2600/118 (20130101) |
Current International
Class: |
C07H
21/04 (20060101); C12Q 1/6883 (20180101); B01D
15/08 (20060101); C12Q 1/68 (20180101); G01N
33/68 (20060101) |
Other References
Woong-Jang (An association between depression after acute coronary
artery syndrome and and methylenetetrahydrofolate reductase gene
polymorphism Feb. 2008, Thesis. Provided on IDS). (Year: 2008).
cited by examiner .
Nashaat et al. (Egyptian J. of Psychiatry, vol. 33, pp. 83-89,
2012). (Year: 2012). cited by examiner .
Tang et al. (Mol Neurobiol, vol. 53, pp. 2152-2160, May 5, 2015).
(Year: 2015). cited by examiner .
Lee et al. (Stress, Brain and Behavior, vol. 5, 1-52, May 16, 2016,
p. 43). (Year: 2016). cited by examiner .
Kang et al (Oncotarget, vol. 7, No. 42, pp. 69032-69040, Sep. 10,
2016). (Year: 2016). cited by examiner .
English Translation of Woong-Jang Kim Thesis "An Association
between Depression after Acute Coronary Artery Syndrome and
Methylenetetrahydrofolate reductase gene polymorphism" Feb. 2008
(Year: 2008). cited by examiner .
Office action dated Sep. 28, 2017 from Korean Patent Office in a
counterpart Korean Patent Application No. 10-2016-0110860. cited by
applicant .
Ys Lee et al., `Longitudinal Associations of Homocysteine and MTHFR
C677T Polymorphism With Depressive Disorder in Patients With Acute
Coronary Syndrome`, Proceedings of the 23rd Multidisciplinary
International Neuroscience and Biological Psychiatry Conference
"Stress, Brain and Behavior", pp. 43, vol. 5, 2016. cited by
applicant .
Woong-Jang Kim , "An association between depression after acute
coronary artery syndrome and methylenetetrahydrofolate reductase
gene polymorphism", Faculty of Medical Sciences Graduate School
Chonnam National University, Gwangju, Korea, pp. 60, Feb. 2008.
(English translation of Abstract is submitted herewith.). cited by
applicant.
|
Primary Examiner: Goldberg; Jeanine A
Attorney, Agent or Firm: The PL Law Group, PLLC
Claims
What is claimed is:
1. A method for diagnosing and treating a patient with chronic
depression after acute coronary syndrome, comprising: a) measuring
a homocysteine concentration in a blood sample from the patient
with acute coronary syndrome and comparing the measured
homocysteine concentration with the standard concentration of 15.0
umol for 1 L of plasma; b) analyzing the methylenetetrahydrofolate
reductase (MTHFR) genotype at position 677; and c) detecting both a
homocysteine concentration equal to or higher than the standard
concentration and a MTHFR 677TT genotype in the patient; d)
diagnosing the patient with chronic depression; and e)
administering an antidepressant to the patient diagnosed with the
chronic depression thereby treating the chronic depression.
2. The method of claim 1, wherein the chronic depression includes
at least one selected from among incident depressive disorder,
which occurs after an acute phase and is present one year after
occurrence of acute coronary syndrome, and persistent depressive
disorder, which occurs during the acute phase and lasts for one
year or more after occurrence of acute coronary syndrome.
3. The method of claim 2, wherein the analyzing the MTHFR genotype
comprises isolating DNA including a 677.sup.th base of a MTHFR gene
separated from the patient, amplifying the isolated DNA using a
sense primer and an antisense primer, and checking whether the
MTHFR 677TT genotype is present by inspecting the amplified DNA
using a restriction enzyme able to recognize a 677C.fwdarw.T
mutation.
4. The method of claim 1, wherein the analyzing the MTHFR genotype
comprises isolating DNA including a 677.sup.th base of a MTHFR gene
separated from the patient, amplifying the isolated DNA using a
sense primer and an antisense primer, and checking whether the
MTHFR 677TT genotype is present by inspecting the amplified DNA
using a restriction enzyme able to recognize a 677C.fwdarw.T
mutation.
5. The method of claim 1, wherein the antidepressant is
escitalopram.
Description
CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY
The present application claims priority to Korean Patent
Application KR 10-2016-0110860 filed on Aug. 30, 2016 in the Korea
Intellectual Property Office, the disclosures of which are
incorporated herein by reference.
BACKGROUND
1. Technical Field
The present invention relates to a method of diagnosing depression,
and more particularly to a method and kit for diagnosing depression
after acute coronary syndrome, in which the concentration of a
specific biomarker in the blood of a patient with acute coronary
syndrome is measured to predict depression after acute coronary
syndrome (ACS).
2. Description of the Related Art
Homocysteine is an amino acid containing thiol, which is produced
via the demethylation of methionine. Homocysteine is metabolized
through remethylation. During remethylation, homocysteine is
converted into methionine by means of a methionine synthase. As
such, vitamin B12 is used as a cofactor, and
5-methyltetrahydrofolate is used as a methyl group donor.
Remethylation plays a main role in maintaining the concentration of
homocysteine in the blood. Here, 5-methyltetrahydrofolate is
prepared from 5,10-methylenetetrahydrofolate by the action of MTHFR
(methylenetetrahydrofolate reductase). When the function of MTHFR
deteriorates, the amount of the 5-methyltetrahydrofolate cofactor
is decreased, whereby remethylation for converting homocysteine
into methionine occurs less readily, thus causing
hyperhomocysteinemia, in which the blood homocysteine concentration
is increased.
A C677T gene mutation of MTHFR, which is the enzyme for controlling
the blood homocysteine concentration, results from substituting
alanine with valine by converting the 677.sup.th cytosine into
thymine. In particular, the function of MTHFR is known to be
decreased by 67.6% in persons having a MTHFR homozygosity mutation,
namely 677TT genotype, than in persons of the mutation-free normal
677CC genotype. Therefore, the homozygosity mutation of the MTHFR
gene is regarded as a strong factor associated with the onset of
hyperhomocysteinemia.
The homocysteine concentration is significantly affected by genetic
factors governing methyltetrahydrofolate reductase (MTHFR). Since
the 677C.fwdarw.T variant of the MTHFR gene is responsible for
thermolabile MTHFR having decreased enzymatic activity, the T
allele is associated with high homocysteine concentration. In this
regard, the T allele of the MTHFR gene is reported to be associated
with an increase in ACS susceptibility upon meta-analysis, and is
found to be associated with depression in some research.
Specifically, homocysteine may cause atherothrombosis due to a
variety of kinds of damage to the blood vascular system, and thus
the risk of ACS increases, and furthermore, homocysteine directly
suppresses the metabolism of a monoamine neurotransmitter and is
thus associated with depression. However, whether homocysteine is a
typical biomarker for depression in ACS has not yet been
investigated.
SUMMARY
Culminating in the present invention, intensive and thorough
research carried out by the present inventors, aiming to solve the
problems encountered in the related art, resulted in the finding
that blood homocysteine concentration and gene polymorphism are
significantly associated with the onset of depression after acute
coronary syndrome.
Accordingly, the present invention is intended to provide a method
of diagnosing depression after acute coronary syndrome, wherein,
based on the results of measurement of homocysteine concentration
in the blood of patients with acute coronary syndrome and MTHFR
(methylenetetrahydrofolate reductase) gene polymorphism, which has
an influence thereon, not only acute depression after acute
coronary syndrome but also chronic depression, occurring one year
or more subsequent to acute coronary syndrome, may be predicted and
diagnosed.
In addition, the present invention is intended to provide a kit for
diagnosing depression after acute coronary syndrome, wherein, based
on the results of measurement of homocysteine concentration in the
blood of patients with acute coronary syndrome and MTHFR gene
polymorphism, chronic depression, as well as acute depression after
acute coronary syndrome, may be predicted and diagnosed, thus
enabling the preemptive prevention of depression in patients and
exhibiting clinical usefulness.
The aspects of the present invention are not limited to the
foregoing, and other aspects not mentioned herein will be able to
be clearly understood to those skilled in the art.
Therefore, the present invention provides a method of diagnosing
depression after acute coronary syndrome, comprising: measuring a
homocysteine concentration in a blood of a patient with acute
coronary syndrome, and determining whether acute depression occurs
by comparing the measured homocysteine concentration with a
standard concentration.
In a preferred embodiment, the determining comprises diagnosing
acute depression when the measured homocysteine concentration is
equal to or higher than the standard concentration.
In a preferred embodiment, when the measured homocysteine
concentration is equal to or higher than the standard
concentration, a likelihood of acute depression occurring is 60% or
more.
In a preferred embodiment, when the measured homocysteine
concentration is less than the standard concentration, a likelihood
of acute depression not occurring is 60% or more.
In a preferred embodiment, the standard concentration is 15.0
.mu.mol or more for 1 L of plasma.
In addition, the present invention provides a method of diagnosing
depression after acute coronary syndrome, comprising: measuring a
homocysteine concentration in a blood of a patient with acute
coronary syndrome, analyzing a methylenetetrahydrofolate reductase
(MTHFR) genotype by measuring MTHFR gene polymorphism of the
patient, and determining whether chronic depression occurs based on
the measured homocysteine concentration and the analyzed MTHFR
genotype.
In a preferred embodiment, in the determining, when both a first
condition, in which the measured homocysteine concentration is
equal to or higher than a standard concentration, and a second
condition, in which the analyzed MTHFR genotype of the patient is a
MTHFR 677TT genotype, are satisfied, chronic depression is
determined to occur.
In a preferred embodiment, the chronic depression includes at least
one selected from among incident depressive disorder, which occurs
after an acute phase and is present one year after occurrence of
acute coronary syndrome, and persistent depressive disorder, which
occurs during the acute phase and lasts for one year or more after
occurrence of acute coronary syndrome.
In a preferred embodiment, when the first condition and the second
condition are satisfied, a likelihood of incident depressive
disorder occurring is 39%, and a likelihood of persistent
depressive disorder occurring is 70% or more.
In a preferred embodiment, when the first condition and the second
condition are not satisfied, a likelihood of incident depressive
disorder not occurring is 96%, and a likelihood of persistent
depressive disorder not occurring is 73%.
In a preferred embodiment, the standard concentration is 15.0
.mu.mol or more for 1 L of plasma.
In a preferred embodiment, the analyzing the MTHFR genotype
comprises isolating DNA including the 677.sup.th base of a MTHFR
gene separated from the patient, amplifying the isolated DNA using
a sense primer and an antisense primer, and checking whether the
MTHFR 677TT genotype is present by inspecting the amplified DNA
using a restriction enzyme able to recognize a 677C.fwdarw.T
mutation.
In addition, the present invention provides a kit for diagnosing
depression after acute coronary syndrome, comprising a measurement
unit for measuring a plasma homocysteine concentration in the blood
of a patient.
In a preferred embodiment, the kit further comprises a genotype
analysis unit for checking whether a 677TT genotype is contained by
measuring a MTHFR gene polymorphism of the patient.
The present invention has the following superior effects.
In the method of diagnosing depression after acute coronary
syndrome according to the present invention, based on the results
of measurement of the homocysteine concentration in the blood of
patients with acute coronary syndrome and MTHFR
(methylenetetrahydrofolate reductase) gene polymorphism, which has
an influence thereon, not only acute depression after acute
coronary syndrome but also chronic depression occurring one year or
more subsequent to acute coronary syndrome can be predicted and
diagnosed.
In the kit for diagnosing depression after acute coronary syndrome
according to the present invention, based on the results of
measurement of the homocysteine concentration in the blood of
patients with acute coronary syndrome and MTHFR gene polymorphism,
chronic depression, as well as acute depression after acute
coronary syndrome, can be predicted and diagnosed, thus enabling
the preemptive prevention of depression in patients and exhibiting
clinical usefulness.
The aforementioned technical effects of the present invention are
not limited to the foregoing, and the effects of the invention that
can be recognized by those skilled in the art from the following
detailed description even if not explicitly mentioned are also
included herein.
BRIEF DESCRIPTION OF THE DRAWING
The above and other features and advantages of the present
invention will be more clearly understood from the following
detailed description taken in conjunction with the accompanying
drawings, in which:
FIG. 1 shows plasma homocysteine concentrations by depressive
disorder status and MTHFR genotype; and
FIG. 2 shows depressive disorder status by hyperhomocysteinemia and
MTHFR genotype.
DETAILED DESCRIPTION
The terms used herein are merely undertaken to explain specific
examples and not to limit the present invention. Unless otherwise
stated, the singular expression includes a plural expression. In
this application, the terms "include" or "have" are used to
designate the presence of features, numbers, steps, operations,
elements, parts or combinations thereof described in the
specification, and should be understood as not excluding the
presence or additional possibility of one or more different
features, numbers, steps, operations, elements, parts or
combinations thereof.
As used herein, the terms "first", "second", etc. may be used to
describe various elements, but these elements should not be limited
by these terms. These terms are only used to distinguish one
element from another element. For example, the first element may be
referred to as a second element or the second element may be
referred to as a first element, within the scope of the present
invention.
Unless otherwise defined, all of technical or scientific terms used
herein have the same meanings as those typically understood by
persons having ordinary knowledge in the art to which the present
invention belongs.
In interpreting the elements, an error range is to be construed as
being included, even if there is no separate description
thereof.
For a description of temporal relationships, when the temporal
posterior relationship, such as "after", "subsequent to",
"following", "before" and so on, is explained, non-continuous cases
are also included, so long as "immediately" or "directly" is not
used.
The characteristic portions of embodiments of the present invention
may be partially or wholly coupled or combined with each other, and
a variety of technical operations thereof are possible, and thus
the embodiments may be independently or jointly performed.
Hereinafter, a detailed description will be given of preferred
embodiments of the present invention with reference to the
accompanying drawings.
However, the present invention is not limited to the embodiments
described herein but may be embodied into different forms.
Throughout the specification, the same reference numerals will
refer to the same or like elements.
The present invention addresses a method and kit for diagnosing
depression after acute coronary syndrome, wherein the blood
homocysteine concentration and MTHFR gene polymorphism are found to
be significantly associated with depression after acute coronary
syndrome and are thus used as biomarkers in order to predict and/or
diagnose not only acute depression but also chronic depression
after acute coronary syndrome.
In the method of diagnosing depression after acute coronary
syndrome according to the present invention, a first diagnosis
method for diagnosing acute depression comprises the steps of
measuring the blood homocysteine concentration of a patient with
acute coronary syndrome and determining whether acute depression
occurs by comparing the measured homocysteine concentration with a
standard concentration, and a second diagnosis method for
diagnosing chronic depression comprises the steps of measuring the
blood homocysteine concentration of a patient with acute coronary
syndrome, analyzing the MTHFR genotype by measuring MTHFR gene
polymorphism in the patient, and determining whether chronic
depression occurs based on the measured homocysteine concentration
and the analyzed MTHFR genotype.
The standard concentration may be 12.0 .mu.mol or more for 1 L of
plasma, and is preferably 15.0 .mu.mol or more for 1 L of plasma,
which is a standard clinical concentration suitable for use in
diagnosing hyperhomocysteinemia.
As used herein, the term "acute phase" refers to a period within
two weeks after occurrence of acute coronary syndrome, and the term
"chronic phase" refers to a period of one year or more after
occurrence of acute coronary syndrome, subsequent to the acute
phase.
Therefore, acute depression indicates depression that occurs within
two weeks after occurrence of acute coronary syndrome, and chronic
depression has no temporal relationship with the time of onset of
depression, so long as it is associated with acute coronary
syndrome, but indicates depression that occurs even one year or
more following acute coronary syndrome. Examples of chronic
depression may include incident depressive disorder, which is
absent during the acute phase but occurs subsequent to the acute
phase and is present one year after occurrence of acute coronary
syndrome, and persistent depressive disorder, which occurs during
the acute stage and lasts for one year or more after occurrence of
acute coronary syndrome.
More specifically, the determination step of the first diagnosis
method may comprise diagnosing acute depression when the measured
homocysteine concentration is equal to or higher than a standard
concentration. Based on statistical results that will be described
later, when the measured homocysteine concentration is equal to or
higher than a standard concentration, the likelihood of acute
depression occurring is 60% or more. On the other hand, when the
measured homocysteine concentration is less than a standard
concentration, the likelihood of acute depression not occurring is
60% or more.
Also, in the determination step of the second diagnosis method,
when both a first condition, in which the measured homocysteine
concentration is equal to or higher than a standard concentration,
and a second condition, in which the analyzed MTHFR genotype of the
patient is the MTHFR 677TT genotype, are satisfied, chronic
depression is determined to occur. Specifically, when the first
condition and the second condition are both satisfied, the
likelihood of incident depressive disorder occurring is 39%, and
the likelihood of persistent depressive disorder occurring is 70%
or more. On the other hand, when the first condition and the second
condition are not satisfied, the likelihood of incident depressive
disorder not occurring 96%, and the likelihood of persistent
depressive disorder not occurring is 73%.
Here, the step of analyzing the genotype may include isolating DNA
including the 677.sup.th base of the MTHFR gene separated from the
patient, amplifying the isolated DNA using a sense primer and an
antisense primer, and checking whether the MTHFR 677TT genotype is
present by inspecting the amplified DNA using a restriction enzyme
able to recognize 677C.fwdarw.T mutation.
In addition, the present invention addresses a kit for diagnosing
the onset of depression after acute coronary syndrome, comprising a
measurement unit for measuring the plasma homocysteine
concentration in the blood of a patient. As necessary, the kit may
further include a genotype analysis unit for checking whether the
677TT genotype is contained by measuring the MTHFR genotype of the
patient.
In this way, the method and kit for diagnosing depression after
acute coronary syndrome according to the present invention are
clinically advantageous because at least one of a high blood
homocysteine concentration of the patient with acute coronary
syndrome and the MTHFR 677TT genotype affects depression after
acute coronary syndrome, and particularly, high blood homocysteine
concentration is significantly associated with acute depression
after acute coronary syndrome, and high blood homocysteine
concentration and MTHFR 677TT genotype are significantly associated
with chronic depression, thereby predicting depression after acute
coronary syndrome and preemptively observing the patient so that
depression, which worsens the prognosis of acute coronary syndrome
patients, may be treated.
EXAMPLES
1. Subject of Study
The data of the present research were deduced for analysis from
larger study named the Korean DEPression in ACS (K-DEPACS) study.
The K-DEPACS study was carried out from 2006 to investigate the
epidemiology of depression in ACS using observational prospective
design. All of the participants were consecutively recruited from
patients (N=4809) who were recently hospitalized with ACS at the
Department Of Cardiology, Chonnam National University Hospital. The
patients were treated based on international guidelines for the
management of ACS by the cardiologists who participated in the
study. The participants (N=1152) who met the eligibility criteria
and agreed to participate in this study were assessed for a
depressive disorder diagnosis by the study psychiatrists using the
Mini-International Neuropsychiatric Interview (MINI) as in patients
within 2 weeks post-ACS, and thereafter as outpatients at intervals
of four weeks up to 12 weeks. Among them, 969 patients agreed to
blood assay, and comprised the baseline sample. Among 378 patients
with depressive disorder in the samples, 255 patients agreed to
participate in a 24 week, double-blind and randomized
placebo-controlled trial to evaluate the efficacy and safety of
escitalopram: EsDEPACS study (Clinical Trial. gov Registry No.:
NCT00419471). The first patient was enrolled at May, 2007, and the
last patient completed the follow-up evaluation at March, 2013. The
evaluation was performed at baseline, 4 weeks, 8 weeks, 12 weeks,
16 weeks, 20 weeks and 24 weeks. Based on the details and main
results of this test, escitalopram was found to be superior to
placebo in reducing depressive symptoms. The remaining 123 patients
who refused to participate in the study were subjected only to
conventional ACS medical treatment. All the participants in
K-DEPACS and EsDEPACS were approached for a re-examination of
depressive status at one year after evaluation at baseline (within
two weeks after ACS). Written informed consent was collected for
the K-DEPACS and EsDEPACS studies, both of which were approved by
the Chonnam National University Hospital Institutional Review
Board.
2. Evaluation of Depressive Status
The diagnosis of depressive disorder was performed using the MINI,
a structured diagnostic psychiatric interview for DSM-IV, which
defines major or minor depressive disorder. The symptom-duration
criteria at baseline was within two weeks. Since the number of
patients with major depressive disorder was not enough to merit
additional analysis, depressive disorder was defined as the
combined category of major depressive disorder and minor depressive
disorder. Based on the evaluation at two phases (two weeks and one
year) after ACS, depressive disorder was classified into acute
depression (prevalent depressive disorder), in which depressive
disorder was present at baseline, and chronic depression, in which
depression was present one year after occurrence of ACS, regardless
of the time of onset of depression [incident depressive disorder:
cases subsequently occurring in the sample without depressive
disorder at baseline, or persistent depressive disorder: cases
subsequently occurring in the sample with depressive disorder at
baseline].
3. Measurement of Plasma Homocysteine Concentration
Blood was collected from a vein in the upper aim of the patient
before breakfast after fasting for 8 hr prior to blood sampling.
The whole blood was placed in a vacutainer tube containing
trisodium EDTA, cooled with ice, and immediately centrifuged at
2,000 rpm for 5 min to separate plasma, and the collected plasma
was stored within 2 hr at -80.degree. C. The total plasma
homocysteine concentration was measured using commercially
available high-performance liquid chromatography (AxSYM
Homocysteine Reagent Pack Abbott, USA).
4. Analysis of MTHFR C677T Polymorphism
DNA isolation was performed from the leukocyte of the blood of a
patient using a DNA extraction kit (extraction column, QIAmp blood
kit, Qiagen) according to the manufacturer's protocol. The isolated
DNA specimen was amplified using a primer set comprising a sense
primer (5'-TGAAGGAGAAGGTGTCTGCGGGA(SEQ ID NO 1)-3') and an
antisense primer (5'-AGGACGGTGCGGTGAGAGTG(SEQ ID NO 2)-3') by means
of a GeneAmp PCR machine (Perkin Elmer 9600). In order to amplify
the 198 bp product obtained through the above PCR amplification, a
series of processes of denaturation at 95.degree. C. for 60 sec,
primer annealing at 62.degree. C. for 90 sec, and primer extension
at 72.degree. C. for 60 sec was repeated for 35 cycles. The
amplified fragments were digested at 37.degree. C. for 4 hr with a
restriction enzyme Hin f1 (10 unit/reaction mixture, available from
MBI Fermentas) able to recognize the 677C.fwdarw.T mutation.
Thereafter, the Hin f1-treated fragments were electrolyzed with
polyacryl amide gel and then stained with EtBr (ethidium bromide)
to observe the mutation status.
5. Demographic and Clinical Covariates
The characteristics of factors that confound or mediate the
association between depressive disorder and ACS were evaluated at
baseline. The data for age, gender, education, living status
(living alone or not), housing (own or rent), current occupation
(currently employed or not), and previous and family histories of
depression were obtained. The following cardiovascular risk factors
were checked: previous and family histories of ACS, diagnosed
hypertension and diabetes, hypercholesterolemia based on fasting
serum total cholesterol level (200 mg/dL), obesity measured by body
mass index (BMI), and current smoking habits. In order to measure
the current cardiac status, the severity of ACS was estimated by
the Killip classification, the left ventricular ejection fraction
(LVEF) was estimated using echocardiography, and serum cardiac
biomarkers, such as troponin I and creatine kinase-MB (CK-MB), were
measured. Other factors able to affect the homocysteine
concentration, such as serum creatinine level and vitamin
supplement, were taken into consideration.
6. Statistical Analysis
Demographic and clinical characteristics were compared between
patients with and without depressive disorder at baseline using
t-tests or chi-squared (.chi..sup.2) tests, as appropriate. The
characteristics significantly associated with depressive disorder
(p<0.05) were used as covariates in other regression models. In
the first analysis, plasma homocysteine was regarded as a
continuous value. The homocysteine concentration and the MTHFR
C677T gene polymorphism were compared between patients with and
without prevalent/incident/persistent depressive disorder using
t-tests and .chi..sup.2 tests, respectively. Odds ratios (ORs) for
depressive status were estimated using a logistic regression model
after adjustment for relevant covariates. In order to evaluate the
effects of potential interactions of homocysteine concentration and
MTHFR C677T polymorphism on depressive status, the following
analyses were performed: i) the homocysteine concentration values
were compared between genotypes using ANOVA; ii) the association
between homocysteine concentration and depressive status was
assayed in each genotype; and iii) the bidirectional interaction
between homocysteine concentration and genotype was tested using a
multivariate regression model. Additional analysis was performed in
the same manner as above in order to investigate the association of
clinically significant categories of hyperhomocysteinemia, defined
by a plasma level of 15.0 .mu.mol/L or more. Also, diagnostic
statistics, for example, sensitivity, specificity, positive
predictive value (PPV) and negative predictive value (NPV), were
calculated for the status of depressive disorder. Also, sensitivity
analysis was performed in the same manner as above under the
condition that participants with a previous history of depression
were excluded. The statistical analysis was conducted using SPSS
18.0 software.
7. Results
(1) Demographic and Clinical Characteristics of All
Participants
Among 969 participants, 378 participants suffered from prevalent
depressive disorder (major or minor) [39.0%; major and minor
depressive disorder were 177 (18.3%) and 201 (20.7%),
respectively]. The baseline demographic and clinical
characteristics affecting prevalent depressive disorder were shown
in Table 1 below. Prevalent depressive disorder (depressive
disorder at baseline) was significantly associated with females,
low education, living alone, rental housing, currently unemployed,
high HAMD score, the presence of hypertension and diabetes, and
current smoking habits.
TABLE-US-00001 TABLE 1 No depressive Depressive disorder disorder
Statistical (N = 591) (N = 378) coefficient p-value*
Socio-demographic characteristics Age, mean (SD) years 57.7 (11.3)
59.0 (10.8) t = -1.860 0.063 Gender, N (%) female 118 (20.0) 151
(39.9) .chi..sup.2 = 45.90 <0.001 Education, mean (SD) years
10.2 (4.8) 9.3 (4.4) t = +3.013 0.003 Living alone, N (%) 47 (8.0)
45 (11.9) .chi..sup.2 = 4.191 0.041 Housing, N (%) rented 73 (12.4)
77 (20.4) .chi..sup.2 = 11.33 0.001 Currently unemployed, N (%) 192
(32.5) 176 (46.6) .chi..sup.2 = 19.39 <0.001 Depression
characteristics Previous depression, N (%) 17 (2.9) 17 (4.5)
.chi..sup.2 = 1.789 0.181 Family history of depression, N (%) 11
(1.9) 12 (3.2) .chi..sup.2 = 1.716 0.190 HAMD, mean (SD) score 3.6
(2.7) 14.2 (5.0) t = -38.09 <0.001 Cardiac risk factors, N (%)
Previous ACS 20 (3.4) 19 (5.0) .chi..sup.2 = 1.610 0.205 Family
history of ACS 15 (2.5) 16 (4.2) .chi..sup.2 = 2.138 0.144
Hypertension 252 (42.6) 206 (54.5) .chi..sup.2 = 13.01 <0.001
Diabetes mellitus 93 (15.7) 98 (25.9) .chi..sup.2 = 15.13 <0.001
Hypercholesterolemia 296 (50.1) 190 (50.3) .chi..sup.2 = 0.003
0.956 Obesity 259 (43.8) 156 (41.3) .chi..sup.2 = 0.614 0.433
Current smoker 249 (42.1) 117 (31.0) .chi..sup.2 = 12.26 <0.001
Current cardiac status Killip class >1, N (%) 97 (16.4) 71
(18.8) .chi..sup.2 = 0.904 0.342 LVEF, mean (SD) % 61.4 (11.2) 60.8
(11.4) t = +0.772 0.440 Troponin I, mean (SD) mg/dL 9.9 (16.6) 9.9
(11.8) t = +0.063 0.949 CK-MB, mean (SD) mg/dL 17.6 (41.1) 17.1
(30.4) t = +0.178 0.858 Other factors Creatinine, mean (SD) mg/dl
0.9 (0.3) 0.9 (0.3) t = +1.013 0.462 Vitamin supplement, N (%) 13
(2.2) 10 (2.6) .chi..sup.2 = 0.198 0.657 *p-values using t-tests or
.chi..sup.2 tests as appropriate. HAMD, Hamilton Depression Rating
Scale; ACS, acute coronary syndrome; LVEF, left ventricular
ejection fraction; CK-MB, Creatine kinase-MB.
(2) Analysis of Association of Homocysteine Concentration and MTHFR
Gene Polymorphism with Status of Depressive Disorder at Baseline
and at Follow-Up
Among all 969 participants at baseline, 711 participants (73.4%)
were successfully followed. The remaining 258 participants lost to
follow up, were older and had higher Killip class (p<0.05) than
the participants who were subjected to follow-up observation. Among
the 426 participants not suffering from depressive disorder at
baseline, the number of patients with incident depressive disorder,
i.e. who were observed to have depressive disorder at follow-up was
53 (12%), and 130 patients (46%) among the 285 participants
suffering from depressive disorder at baseline were observed to
have persistent depressive disorder, in which depressive disorder
was still present even at follow-up.
In order to analyze the association of homocysteine concentration
and MTHFR gene polymorphism with the status of acute depression and
chronic depression, the plasma homocysteine concentration and MTHFR
gene polymorphism were analyzed from the blood of ACS patients only
at baseline. The depressive status of patients based on the
evaluation at baseline and follow-up was classified into acute
depression, that is, prevalent depressive disorder, and chronic
depression, that is, incident depressive disorder and persistent
depressive disorder, and, depending on the status of each kind of
depressive disorder, the association of the analyzed MTHFR genotype
and the measured plasma homocysteine concentration were analyzed.
The results are shown in FIG. 1 and Tables 2 and 3 below.
With reference to FIG. 1, the numeral data indicate plasma
homocysteine concentration (.mu.mol/L). Depending on the presence
or absence of depressive disorder, p-values were deduced using a
logistic regression test after adjustment for gender, education,
living status, housing type, current employment status,
hypertension, diabetes, current smoking habits (prevalent
depressive disorder and incident depressive disorder) and treatment
status (persistent depressive disorder). The interaction between
the homocysteine concentration and the MTHFR genotype on the
depressive status was deduced from the same adjusted model. In
Tables 2 and 3, p-values resulted from t-tests or .chi..sup.2
tests.
TABLE-US-00002 TABLE 2 Prevalent depressive Incident depressive
Persistent depressive disorder at 2 weeks disorder at 1 year
disorder at 1 year after ACS after ACS after ACS Absent Present
Absent Present p- Absent Present p- (N = 591) (N = 378) p-value (N
= 373) (N = 53) value (N = 155) (N = 130) value Homocysteine, 10.2
12.0 <0.001 10.0 10.7 0.269 11.7 12.6 0.157 mean (SD) (3.7)
(5.1) (3.7) (4.3) (4.8) (5.9) .mu.mol/l MTHFR genotype, N (%) C/C
214 128 0.749 136 18 0.740 51 41 0.882 (36.2) (33.9) (36.5) (34.0)
(32.9) (31.5) C/T 277 185 177 28 73 65 (46.9) (48.9) (47.5) (52.8)
(47.1) (50.0) T/T 100 65 60 7 31 24 (16.9) (17.2) (16.1) (13.2)
(20.0) (18.5)
TABLE-US-00003 TABLE 3 Homocysteine Homocysteine concentration X
concentration MTHFR genotype MTHFR genotype Wald OR (95% CI) Wald
OR (95% CI) Wald OR (95% CI) Prevalent depressive 30.63 1.11
(1.07-1.15)*** 0.14 0.96 (0.79-1.18) 1.19 1.03 disorder.sup.a
(0.98-1.08) Incident depressive 0.51 1.03 (0.95-1.11) 0.05 0.95
(0.62-1.47) 3.65 1.10 disorder.sup.a (1.00-1.20)* Persistent
depressive 1.13 1.04 (0.99-1.09) 0.17 0.93 (0.65-1.32) 3.90 1.12
disorder.sup.b (1.02-1.23)* .sup.aadjusted for gender, education,
living alone, housing, current employment, hypertension, diabetes,
and current smoking .sup.badjusted for the same model as in
prevalent depressive disorder plus treatment status (escitalopram,
placebo, and medical treatment only) *p-value <0.05, **p-value
<0.01, ***p-value <0.001
As is apparent from FIG. 1 and Tables 2 and 3, considerably high
homocysteine concentration was significantly associated with acute
depression, namely prevalent depressive disorder, but was not
significantly associated with chronic depression, namely incident
depressive disorder and persistent depressive disorder. In the case
of the MTHFR genotype, no deviation from Hardy-Weinberg equilibrium
was observed (p-value=0.673). Also, no significant associations
were found for MTHFR genotype with any depressive disorder (all
p-values >0.7). In order to evaluate whether the homocysteine
concentration and the MTHFR genotype are associated with depressive
status, logistic regression analysis was performed after adjustment
for covariates, whereby similar results were obtained.
As for the interaction of homocysteine concentration and MTHFR
genotype depending on the depressive status, FIG. 1 shows the
results of comparison of homocysteine concentrations depending on
the status of depressive disorder in each genotype. The higher
homocysteine concentration was associated with prevalent depressive
disorder in the presence of the C/T and T/T genotypes, and with
incident depressive disorder and persistent depressive disorder
only in the presence of the T/T genotype. The interactions between
genotype and raised homocysteine in logistic regression models
after adjustment for covariates are summarized in Table 3. Although
an important interaction between homocysteine concentration and the
MTHFR genotype was found in incident depressive disorder and
persistent depressive disorder, it was not found in prevalent
depressive disorder.
(3) Analysis of Association of Clinical Hyperhomocysteinemia and
MTHFR Gene Polymorphism with the Status of Depressive Disorder
The prevalence rate of hyperhomocysteinemia was 16.1% (156 of 969
participants), and association with depressive status is
illustrated in FIG. 2. In all samples, hyperhomocysteinemia was
significantly associated with prevalent depressive disorder
(sensitivity of 64%, and specificity of 63%) and incident
depressive disorder, but not with persistent depressive disorder.
Upon analysis based on the MTHFR genotype, hyperhomocysteinemia was
significantly associated with prevalent depressive disorder in the
presence of C/T and T/T genotypes, and hyperhomocysteinemia was
associated with incident depressive disorder and persistent
depressive disorder in the presence of the T/T genotype (PPV and
NPV for incident depressive disorder were 39% and 96%,
respectively, and PPV and NPV for persistent depressive disorder
were 78% and 73%, respectively). Based on the results of analysis
of logistic regression after adjustment for covariates, an
important interaction between hyperhomocysteinemia and genotype was
found in incident depressive disorder (p-value=0.030) and
persistent depressive disorder (p-value=0.027), but was not found
in prevalent depressive disorder (p-value=0.243).
Such test results support a crucial role of homocysteine in the
pathogenesis of depressive disorder comorbid with ACS independently
at acute phase and interactively with MTHFR genotype at chronic
phase. Thus, plasma homocysteine analysis, especially clinical
hyperhomocysteinemia analysis, makes it possible to screen the risk
group of depressive disorder in the acute phase of ACS, whereby
appropriate treatment may be applied and which is thus clinically
useful.
Furthermore, depression in ACS leads to a very high disease burden
and is difficult to treat. According to the present invention,
assaying homocysteine and MTHFR genotype may allow more focused
interventions for the prevention of management of depressive
disorder in the susceptible sub-group for depressive disorder not
only in the acute phase of ACS but also in the chronic phase of
ACS. Therefore, the present invention is useful in the prevention
or preemptive treatment of early-stage depression after ACS and/or
before the onset of depression.
Although the preferred examples and test examples of the present
invention have been disclosed for illustrative purposes, those
skilled in the art will appreciate that various modifications,
additions and substitutions are possible, without departing from
the scope and spirit of the invention as disclosed in the
accompanying claims.
SEQUENCE LISTINGS
1
2123DNAArtificial Sequencesense primer 1tgaaggagaa ggtgtctgcg gga
23220DNAArtificial Sequenceantisense primer 2aggacggtgc ggtgagagtg
20
* * * * *